The Role Of BAP1/KLF5 Regulating FGF-BP1/Snail2 Signaling Axis In Progression Of Esophageal Squamous Carcinoma

Esophageal cancer(EC)is one of the most common malignant tumors in China.EC is classified as esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC),with the latter accounting for over 90%of all esophageal tumors.Due to the insidious onset of esophageal cancer,the early symptoms are not obvious.Most patients are diagnosed at the late stages,and present with distant metastasis or local infiltration of the tumor.Systemic chemotherapy and radiotherapy are the main treatment options for metastatic ESCC.The overall 5-year survival rate is low.In recent years,targeted therapy has shown promising effects in several malignant tumors,but for esophageal squamous cell carcinoma,clinical management of ESCC remains challenging and the disease presently lacks approved targeted therapeutics.Therefore,it is necessary to explore the regulation mechanism of esophageal squamous cell carcinoma development and progression to identify candidate biomarkers for ESCC.Kruppel-like factor 5(Kruppel-like factor 5,KLF5)is one of the key members in KLF family.Under physiological conditions,KLF5 extensively controls several important cellular processes,including cell proliferation,differentiation,movement,inflammation and pluripotency.However,KLF5 deregulation has been proven to be a transcriptional activator or inhibitor,which affects the regulation of gene expression in normal cells and intervenes cell proliferation,cell cycle,survival,angiogenesis and stem cell self-renewal,eventually leading to the tumor formation.Therefore,KLF5 can be considered as a potential molecular marker and therapeutic target.At present,the potential role of KLF5 in esophageal cancer has not been fully discussed.A previous study revealed that KLF5 is an unstable protein,and posttranslational modifications of KLF5,including ubiquitination,SUMOylation,acetylation and phosphorylation can impact both the stability and activity of KLF5,thus affecting its downstream cellular functions.Among them,the ubiquitinproteasome pathway(UPP)plays a key role in the process which tightly control KLF5 level.The dynamic balance between protein degradation and deubiquitination caused by ubiquitination plays an important role in KLF5 regulation.Therefore,we speculate that KLF5 protein level in esophageal squamous cell carcinoma is not only regulated by KLF5 mRNA transcription and translation,but also by post-translational modification.Previous studies showed that the deubiquitinating enzyme BAP1 could increase the stability of KLF5 protein through deubiquitination in triple-negative breast cancer,and up-regulate TNFAIP2 expression to promote breast cancer proliferation,invasion and metastasis,while knocking down BAP1 or KLF5 can inhibit triple-negative breast cancer cell growth and lung metastasis in nude mice,suggesting that BAP1 may affect KLF5 expression through deubiquitination pathways and targeted binding dual pathways,but whether BAP1 is an upstream regulator for KLF5 in esophageal squamous cell carcinoma need to be confirmed.In this study,we first detected the expression of KLF5 in esophageal squamous cell carcinoma tissues and cell lines,and the effects of KLF5 on the proliferation,migration and invasion of esophageal squamous cell carcinoma cells were also analyzed.Finally,the BAP1 and FGF-BP1 signaling pathway was investigated as the mechanism underlying the effects of KLF5 in esophageal squamous cell carcinoma cells.The specific functions and mechanisms in the process are expected to provide new potential targets for the clinical treatment of esophageal squamous cell carcinoma based on the determination of the BAP1/KLF5 signal axis.Part Ⅰ KLF5 Expression and clinical significance in ESCCObjective:UALCAN bioinformatic analyses and IHC evaluation were conducted to evaluate KLF5 expression in ESCC,and the correlation between its expression and clinicopathological characteristics in patients were analyzed.Methods:(1)We conduct a comprehensive bioinformatics analysis based on ESCC data in UALCAN databases to evaluate the expression of KLF5 in squamous cell carcinoma..(2)ESCC specimens were collected,and KLF5 protein expression were detected by immunohistochemistry.For the analysis of KLF5,the data were divided into low expression group,medium and high expression group by comprehensively judging by staining intensity and staining positive rate;chi-square test were used to evaluate the correlation between KLF5 protein expression and clinicopathological characteristics in ESCC.Results:1.Bioinformatics analysis of KLF5 mRNA expression in ESCC tissuesSignificant upregulation of KLF5 mRNA expression were found in ESCC tissues as compared with control group(p<0.05).2.Bioinformatics analysis of the relationship between KLF5 expression and the prognosis in ESCCKaplan-Meier survival analysis showed that there was no correlation between KLF5 expression and OS in ESCC patients.3.KLF5 protein expression in ESCCAmong the 47 ESCC samples,the protein expression of KLF5 were detected in 37 ESCC tissues by IHC.The staining was observed in both cellular cytoplasm and nucleus.Twenty-one 21 samples(44.7%)had no or little KLF5 expression(low group,scores 0,1+),whereas 26 patients(55.3%)exhibited a strong staining(high group,scores 2+,3+).4.Correlation between KLF5 expression and clinicopathological parameters in ESCCThe chi-square test analysis demonstrated that up-regulation of KLF5 expression was significantly associated with ESCC aggressive tumor phenotypes,such as poor differentiation(p=0.0012)and more lymph node metastasis(p=0.037).There was no significant correlation between KLF5 expression and age(p=0.970),gender(p=0.529),tumor location(p=0.621)and pathological grade(p=0.134).Conclusions:KLF5 protein may play an oncogene role in the progression of esophageal squamous cell carcinoma,and the elevated KLF5 level were closely related with aggressive tumor phenotypes,including poor differentiation and the presence of distant metastases.Part Ⅱ Exploration the regulation effects of BAP1 on KLF5Objective:To analyze the expression level of BAP 1 in esophageal squamous cell carcinoma,and to explore the effects of BAP1 overexpression and knockdown on the biological function of esophageal squamous cell carcinoma cells;to reveal the regulatory mechanism of BAP 1 on KLF5 and its downstream pathway.Methods:BAP1 protein expression were detected by immunohistochemistry in ESCC specimens.ESCC cells with BAP1 overexpression or inhibition were constructed.CCK8 were used to evaluate the effects of BAP1 on ESCC cell proliferation.Transwell and scratch experiments were used to compare the effects of BAP1 on ESCC cell invasion and migration.The correlations between BAP1 and KLF5 were verified by Western-blotting,rescue experiment,double Luciferase Report and Co-IP.The deubiquitinase inhibitor PR619 and proteasome inhibitor MG 132 were used to reveal whether BAP1 stabilizes KLF5 protein through deubiquitination.Results:1.BAP1 protein expression in ESCCAmong the 47 ESCC samples,the protein expression of BAP1 were detected in 34 ESCC tissues by IHC.The staining was observed in both cellular cytoplasm and nucleus,and mainly detected in nucleus.Eleven samples(23.4%)had no or little BAP1 expression(low group,scores 0,1+),whereas 23 patients(48.9%)exhibited a strong staining(high group,scores 2+,3+).2.Overexpression of BAP1 enhanced cell proliferation and migration in ESCCESCC cells were transfected with pcDNA-BAP1 or an empty vector to induce the overexpression of BAP1.The efficiency of transfection results for pcDNA-BAP1 showed that the expression of BAP1 was increased compared with that of the control.Western blot analysis of the protein expression level of BAP1 confirmed the overexpression of BAP 1 in the pcDNA-BAP1 group.The CCK8 was used to evaluate cell proliferation,and the results revealed that cell proliferation was notably enhanced in ESCC cells transfected with pcDNA-BAP1 compared to those transfected with the empty vector.Cell migration was then evaluated by the wound-healing assay,and the results suggested that the migration of ESCC cells transfected with pcDNA-BAP1 increased notably compared with that in the control.3.Knockdown of BAP1 impeded cell proliferation and migration in ESCC cellsTo further examine the effect of BAP1 on cell proliferation and migration,knockdown of BAP 1 was achieved using siRNA-BAP1.The efficiency of transfection results for siRNA-BAP1 showed that the expression of BAP1 was reduced in the siRNA-BAP1 group compared with the NC group.Similar to the overexpression of BAP1,cell proliferation and migration were measured by CCK8 and wound healing assays in the BAP1 knockdown system.The results showed that cell proliferation and migration in ESCC cells transfected with siRNA-BAP1 were notably inhibited compared with that in the NC group.4.BAP1 expression affected KLF5 and its downstream genes FGF-BP1As expected,the expression of KLF5 was enhanced in the BAP1 overexpression system compared with that in the control.KLF5 mRNA levels were also significantly reduced in ESCC cells transfected with siRNA-BAP1 compared with those in the NC group.Interestingly,we found that the expression of downstream genes,KLF5,and FGF-BP1 and Cyclin D1,were positively regulated by the expression of KLF5.The expression of FGF-BP1 increased or reduced significantly in the BAP1 overexpression or knockdown systems,respectively.In addition,the expression of KLF5,and FGF-BP1 was further verified at the protein level and showed similar tendencies to the mRNA expression levels.5.The "rescue experiment" verified the regulation effects of BAP1 on KLF5"Rescue experiment" were used to explore the regulation effects of BAP1 on KLF5 in ESCC cells by co-transfecting BAP1 and KLF5.The results showed that the proliferation activity was significantly decreased in BAP1+siKLF5 group,as compared with BAP 1+siNC group.6.BAP1 interacts with KLF5The transcriptional activity of KLF5 was detected using dual luciferase reporter gene.Dual luciferase reports showed that BAP1 could not directly bind to the promoter region of KLF5 to promote its transcriptional activity,suggesting that BAP1 promoted KLF5 transcription through an indirect pathway.To further reveal whether BAP1 and KLF5 could interact at the protein level,protein complexes were enriched in protein lysates of wild-type Eca109 cells with BAP1 antibody and KLF5 antibody,respectively,and then verified by western blot.The results show that BAP1 can form a protein complex with KLF5 under physiological conditions.7.PR619 and MG132 intervention experiment assisted to verify the deubiquitination of BAP1 on KLF5In order to clarify the deubiquitination effect of BAP1 on KLF5,we designed PR619 and MG 132 intervention experiment by co-transfecting BAP1,KLF5 and PR619 or MG132.Western-blot was used to detect the effect of BAP1 on KLF5 protein expression.The results show that the protein expression of BAP1 and KLF5 was decreased in the BAP1+PR619 group,but MG132 had no influence on BAP1 and KLF5 expression.Conclusions:BAP1 might promote cell proliferation and migration via maintaining KLF5 protein expression in ESCC cells.Part Ⅲ Exploration the biological role of KLF5 in ESCCObjective:ESCC cells with over-expression or knock-down of KLF5 were constructed to explore its effect on the proliferation,migration and invasion in ESCC.The regulation effects of KLF5 on FGF-BP1-Snail2 pathway were discussed.Methods:ESCC cells with KLF5 overexpression or inhibition were constructed.CCK8 were used to evaluate the effects of KLF5 on ESCC cell proliferation.Transwell and scratch experiments were used to compare the effects of KLF5 on ESCC cell invasion and migration.The correlations between KLF5,FGF-BP1 and Snail2 were analyzed based on GEO datasets,and verified by Western-blotting,ChIP-qPCR and luciferase report assay.Results:1.The effects of KLF5 overexpression on ESCC biological functionWe chose KYSE-150,KYSE-140,and Eca109 cells to establish KLF5 overexpressing cells construction.Western-blotting analysis showed that KLF5 expression were significant higher in treatment group as compared with control group(p<0.05);Cell proliferation was significantly stronger in the pcDNA-KLF5 group in KYSE-150,KYSE-140,and Eca109 cells than that in the blank control group at 48 and 72 h post-transfection(p<0.05);The results of wound healing assay and transwell showed that KLF5 overexpression can enhance the invasion and migration ability in ESCC cells(p<0.05).2.The effects of KLF5 knock-down on ESCC biological functionESCC cell lines presented the knockdown of KLF5,when plasmid siKLF5 was transfected.Cell proliferation was significantly weaker in the siKLF5 group in three ESCC cell lines than that in the siNC at 48 or 72 h post-transfection.KLF5-reduced ESCC cells revealed significantly decreased invasion and migration capabilities compared with their corresponding control cells.3.KLF5 promoteds ESCC invasion and metastasis via activating FGF-BP1Snail2 signalThe above results suggested the involvement of KLF5 in ESCC cells metastasis.Next,TCGA database were processed to further explore the possible mechanism underlying KLF5-driving ESCC.TCGA analysis revealed a significantly positive correlation association between KLF5 and FGF-BP1 mRNA expression in ESCC specimens.To confirm the interaction between KLF5 and FGF-BP1,two human microarray datasets(GSE44021 and GSE16355 and GSE44021)from GEO were used to evaluate the mRNA expression of KLF5 and FGF-BP1.Likewise,the positive correlation associations between KLF5 and FGF-BP1 were also found in GEO datasets.Western-Blotting revealed the influence of KLF5 on FGF-BP1 and Snail2 expression.The above results indicated that the function of KLF5 may be related to the FGF-BP1.These findings indicated that FGF-BP1 might be implicated in biological functions of KLF5 in ESCC.4.KLF5 binds to the FGF-BP1 promoter region to directly promote its expressionChIP-qPCR confirmed that KLF5 was bound to the FGF-BP1 promoter,but KLF5 was only bound to the predicted site 1,not the other predicted sites.Dual lucifase reporter assay verified that FGF-BP1 was the direct target gene of KLF5 after point mutation in the TGGGTGAGGC region of FGF-BP1 promoter.5.The "rescue experiment" verified the regulation of FGF-BP1 on Snail2 expression."Rescue experiment" were used to explore the regulation effects of FGF-BP1 on Snail2 in ESCC cells by co-transfecting FGF-BP1 and Snail2.The results showed that the proliferation activity was significantly decreased,in FGF-BP1+siSnail2 group as compared with FGF-BP1+siNC group.Conclusions:KLF5 can affect the ESCC invasion and migration by regulating FGF-BP1-Snail 2 signal axis.